RBPs are pivotal regulators of mRNA metabolism, influencing all aspects of post-transcriptional gene control: pre-mRNA splicing, mRNA transport, mRNA stability, and translation. We study if a given RBP associates with a given mRNA by a variety of in vitro binding assays (biotin pulldown, RNA EMSA, surface plasmon resonance/Biacore, etc) and assays to measure binding of endogenous molecules (ribonucleoprotein immunoprecipitation). To investigate RBP function in mammalian cultured cells, we employ approaches such as RBP silencing, RBP overexpression, and the identification of RBP-associated mRNAs using microarrays. We investigate whether RBPs affect the stability of target mRNAs by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs.[unreadable] [unreadable] During this funding period, we have completed two main projects. In one project, we systematically analyzed the interactions of six major RBPs (HuR, AUF1, NF90, KSRP, TIAR, and TIA-1) with the mRNAs that encode these RBPs. We reported that each RBP associates with its own mRNA and uncovered an extensive network of cross-regulation among these RBPs and the mRNAs that encode them. In the other main project, we reported that HuR is phosphorylated by the kinase Cdk1 (Cdc2); Cdk1 phosphorylates HuR at S202 (a residue located within a region important for controlling the subcellular levels of HuR) and together with 14-3-3 proteins, it helped to retain HuR in the nucleus.[unreadable] [unreadable] Ongoing work is examining the subcellular localization of HuR by post-translational modification at S242 (also located within the region that modulates HuR subcellular distribution), and the influence of ubiquitination upon the overall HuR levels in cells responding to heat shock. Other studies are seeking to identify the subsets of mRNAs that are regulated by RBPs NF90 and AUF1.